Enhancer-Associated LncRNA-ITGA2 Promotes Vascular Remodeling Through ITGA2

Abstract

Background: The proliferation and migration of vascular smooth muscle cells (VSMCs) significantly contribute to vascular remodeling. Recent studies have suggested that enhancer-associated long noncoding RNAs (elncRNAs) play crucial roles in regulating gene expression and cell fate. However, the specific elncRNAs implicated in VSMC dysfunction and their regulatory mechanisms remain poorly understood.

Methods: This study used multiomics profiling techniques, including cleavage under targets and tagmentation (CUT&Tag), promoter capture high-throughput chromosome conformation capture, and microarray analysis, to identify long noncoding RNA (LncRNA)-ITGA2 (integrin α2) as a novel elncRNA involved in VSMC dysfunction. A carotid artery wire injury model was used to study its role in vascular remodeling in vivo. RNA sequencing and CRISPR-Cas9 gene-editing technology were employed to explore downstream targets of LncRNA-ITGA2. Chromatin immunoprecipitation sequencing, chromatin isolation by RNA purification, and RNA immunoprecipitation were performed to investigate its mechanistic role in VSMC proliferation and migration.

Results: LncRNA-ITGA2 was highly expressed in PDGF-BB (platelet-derived growth factor BB)-induced proliferative human VSMCs and was elevated in coronary atherosclerotic tissues from coronary artery disease patients compared to controls. Gain-of-function and loss-of-function studies suggested that LncRNA-ITGA2 markedly enhanced PDGF-BB-induced VSMC proliferation and migration. In vivo, the overexpression of LncRNA-ITGA2 promoted neointimal hyperplasia, indicating its partial functional conservation. Mechanistically, we identified ITGA2 as a downstream target of LncRNA-ITGA2. Promoter capture high-throughput chromosome conformation capture detected promoter‒enhancer interactions at the ITGA2 locus following PDGF-BB treatment. LncRNA-ITGA2 could directly bind to the enhancer-ITGA2 and led to an increase in H3K27 acetylation in both the enhancer-ITGA2 and the promoter-ITGA2. Additionally, we found that LncRNA-ITGA2 interacted with the DNA-binding protein NONO (non-pou domain containing octamer-binding protein), which also bound to the ITGA2 promoter. Ultimately, the use of knockout cell lines for NONO, LncRNA-ITGA2, and its promoter validated the proposed regulatory mechanism.

Conclusions: This study identifies a novel mechanism by which elncRNA (LncRNA-ITGA2), in a NONO-dependent manner, mediates interactions between enhancer-ITGA2 and promoter-ITGA2, resulting in increased ITGA2 expression and subsequently promoting VSMC proliferation and migration. These findings highlight LncRNA-ITGA2 as an attractive diagnostic and therapeutic target for human proliferative vascular diseases.

Keywords: RNA, long noncoding; cell proliferation; integrins; muscle, smooth, vascular; vascular remodeling.